Skip to main content
Download PDF

Targeting PCSK9, through an innovative cVLP-based vaccine, enhanced the therapeutic activity of a cVLP-HER2 vaccine in a preclinical model of HER2-positive mammary carcinoma

Journal of Translational Medicine volume 23, Article number: 136 (2025) Cite this article

Abstract

Background

HER2-targeted therapies have revolutionized the treatment of HER2-positive breast cancer patients, leading to significant improvements in tumor response rates and survival. However, resistance and incomplete response remain considerable challenges. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a novel therapeutic strategy for the management of dyslipidemia by enhancing the clearance of low-density lipoprotein cholesterol receptors, however recent evidence also shows links between PCSK9 and cancer cells. We present an innovative immunization approach combining capsid virus-like particle (cVLP)-based vaccines against HER2 and PCSK9.

Methods

The therapeutic activity of the combined vaccine was evaluated in female mice challenged with HER2-positive mammary carcinoma cells. Controls included untreated mice and mice treated with cVLP-PCSK9 and cVLP-HER2 as standalone therapies. Antibodies elicited by vaccinations were detected through ELISA immunoassay. The functional activity of the antibodies was tested in 3D-soft agar assay on human HER2 +  +  + trastuzumab sensitive and resistant cells.

Results

Mice vaccinated with cVLP-HER2 + cVLP-PCSK9 displayed tumor regression from the 40th day after cell challenge in 100% of mice remaining tumor-free even 4 months later. In contrast, 83% of mice treated with cVLP-HER2 vaccine alone experienced an initial tumor regression, followed by tumor relapse in 60% of subjects. Untreated mice and mice treated with the cVLP-PCSK9 vaccine alone developed progressive tumors within 1–2 months after cell injection. The combined vaccine approach elicited strong anti-human HER2 antibody responses (reaching 1–2 mg/ml range) comprising multiple immunoglobulins isotypes. cVLP-PCSK9 vaccine elicited anti-PCSK9 antibody responses, resulting in a marked reduction in PCSK9 serum levels. Although the anti-PCSK9 response was reduced when co-administered with cVLP-HER2, it remained significant. Moreover, both cVLP-HER2 + cVLP-PCSK9 and cVLP-HER2 alone induced anti-HER2 antibodies able to inhibit the 3D growth of human HER2 +  +  + BT-474 and trastuzumab-resistant BT-474 C5 cells. Strikingly, antibodies elicited by the combined vaccination were more effective than those elicited by the cVLP-HER2 vaccine alone in the inhibition of trastuzumab-resistant C5 cells.

Conclusions

The results indicate that cVLP-PCSK9 vaccination shows adjuvant activity when combined with cVLP-HER2 vaccine, enhancing its therapeutic efficacy against HER2-positive breast cancer and holding promise in overcoming the challenges posed by resistance and incomplete responses to HER2-targeted therapy.

Background

Breast cancer is the most common and deadly cancer diagnosed in women. As a highly heterogeneous neoplasm, it is characterized by multiple tumoral entities that differ in biological and histological properties due to transcriptomic, epigenetic and genetic changes that are associated with varying clinical findings and treatment responses [1,2,3].

The oncogenic role of the human epidermal growth factor receptor 2 (HER2), exerted by gene mutation, amplification and/or protein overexpression observed in 15–20% of breast cancer and gastric cancer, 10% of bladder cancer and 3% of colorectal cancer, is commonly associated with aggressive behavior and poor prognosis if not treated promptly [4,5,6,7].

The tumorigenic features of HER2 are related to its oversignaling, and such dysregulation confers enhanced growth abilities, migration, metastasization and avoidance of apoptosis in favor of proliferation [8]. In the specific case of breast cancer, alterations in the expression or functionality of HER2 result in the diagnosis of a specific subtype usually characterized by higher histological grade, higher invasiveness and unfavorable prognosis, while giving simultaneously the advantage of a specific oncogene to target, overexpressed only in cancer cells [9].

The advent of monoclonal antibodies (mAbs) targeting HER2, such as trastuzumab and pertuzumab, small tyrosine kinase inhibitors (TKIs) and more recently of antibody–drug conjugates (ADCs), significantly improved patient response rate and survival. Unfortunately, a variable proportion of patients eventually present intrinsic or acquired resistance to these treatments which leads to the disease progression [10,11,12,13,14].

Resistance to anti-HER2 therapies in breast cancer involves multiple complex mechanisms that challenge the efficacy of treatments [15,16,17,18,19]. One major contributor is genetic alterations, such as HER2 mutations, which can drive tumor growth even without HER2 overexpression [17]. The activation of alternative signaling pathways that counteract the effects of HER2-targeted therapies [20, 21], or the interaction between HER2 and insulin-like growth factor-I receptor (IGF-1R), that induces phosphorylation and activates the PI3K pathway, play a significant role in therapy resistance [22, 23]. Additionally, specific isoforms of HER2, such as HER2Δ16, exhibit constitutive activation of downstream signaling, and truncated variants like p95HER2, which lack the extracellular domains targeted by HER2 antibodies, can further compromise therapeutic efficacy. However, the exact role of these isoforms in resistance remains a topic of debate [24,25,26]. Epitope masking might further reduce treatment efficacy by limiting antibody binding [20, 24]. Indeed, lipid rafts were reported to be involved in regulating HER2 signaling and anti-HER2 therapy response. Among other raft-associated proteins, caveolin-1 seems to modulate receptor localization, impairing the sensitivity of cancer cells to targeted therapies [27, 28]. Tumor heterogeneity also poses significant challenges. Variability in HER2 expression within a single tumor can impair the effectiveness of therapies, making targeting all tumor cells more difficult [29,30,31,32]. Finally, immune evasion mechanisms undermine the efficacy of anti-HER2 therapies, which depend on immune-mediated responses. An immunosuppressive tumor microenvironment (TME) can impair these responses, reducing treatment effectiveness [17, 18]. Additionally, genetic polymorphisms in the FcγRIII receptor alter its binding affinity to IgG1, thereby diminishing the antibody-dependent cellular cytotoxicity (ADCC) mediated by trastuzumab [33, 34].

The incomplete tumor eradication by HER2-targeted therapy, and consequently the onset of recurrences or failure of therapeutic regimens, can be potentially overcome by the combination of different monoclonal antibodies or by the combination with other immunomodulatory agents, such as immune checkpoint inhibitors and immune-stimulating antibody conjugates [13, 17, 35,36,37,38,39,40,41].

From an immunological point of view, an active form of immunization, characterized by a polyclonal and prolonged immunological response could be beneficial to employ a wider range of anti-tumor activities exerted by the different immunoglobulins [42,43,44]. Anti-cancer responses with such characteristics can be obtained with cancer vaccines [45,46,47,48,49].

A further strategy to enhance targeted therapy efficiency is to interfere with the high energetic and metabolic needs of cancer cells [50,51,52,53,54]. HER2-positive breast cancer resistance to targeted therapies, among others, was found to be associated with an increase in fatty acid (FA) metabolism [55,56,57,58]. Several preclinical studies have shown that targeting FA pathways can increase anti-HER2 therapy efficacy or restore treatment response both in breast and gastric cancer [57, 59,60,61,62].

The reprogramming of energy metabolism has been identified as a hallmark of cancer and reported as a mechanism of therapy resistance [50, 55]. Cholesterol homeostasis can gain abnormal features during carcinogenesis and cancer spread and has been associated with increased cancer risk [63,64,65,66,67,68,69,70]. From an immunological perspective, high levels of cholesterol inside cells can lead to T-cell exhaustion or prolongation of the resting state [71,72,73,74]. In a tumor microenvironment enriched in cholesterol, myeloid suppressor cells have been reported to exert immune-suppressive activities [75,76,77]. In addition, cholesterol is involved for the formation of immunological synapses, as it may reduce antigen presentation by dendritic cells through the down-regulation of the MHC-I expression and by decreasing their motility [78,79,80].

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is tightly involved in cholesterol homeostasis, in particular to cholesterol absorption through low-density lipoprotein receptor (LDLR) [81]. This soluble zymogen, primarily secreted by hepatocytes, causes LDLR lysosomal degradation, generating an imbalance in the finely regulated metabolic process that is cholesterol homeostasis. Free LDL cholesterol in the bloodstream is an energetic and architectural source that can be taken-up and metabolized by peripheral cancer cells, while contributing to immuno-suppressive responses in the tumor microenvironment [82].

Indeed, recent evidence demonstrates a link between PCSK9 biological activities and various cancers, including hepatocellular carcinoma (HCC), breast cancer, colorectal cancer (CRC), and prostate cancer (PCa). These changes are frequently associated with increased tumor invasiveness and poorer clinical outcomes [64, 82,83,84]. Some of the proposed mechanisms involve PCSK9 in cell proliferation, apoptosis and stemness [84, 85]. Furthermore, PCSK9's well-known function in cholesterol metabolism, particularly its regulation of LDLR expression, may indirectly affect cancer growth and metastasis. Cholesterol plays a crucial role in cellular processes linked to tumor development, and PCSK9-mediated alterations in cholesterol homeostasis could contribute to these effects [86, 87]. Another intriguing aspect is the potential link between PCSK9 and immune responses. Expressed by immune cells such as macrophages and lymphocytes, PCSK9 appears to regulate immune cell function. This activity suggests that PCSK9 may influence the tumor microenvironment (TME) by modulating immune surveillance and the body’s ability to suppress cancer cell growth [87].

Gain of function (GOF) variants of PCSK9 coding gene or high expression levels of PCSK9 were found to be associated with reduced survival of breast cancer patients, particularly HER2-positive and triple-negative ones (https://kmplot.com/analysis/, accessed on 22nd of April, 2024)[83]. Moreover, a common missense germline variant in PCSK9 (V474I) was recently found associated with breast cancer metastasis and reduced survival in multiple breast cancer patient cohorts [88].

Passive immunotherapy against PCSK9, through monoclonal antibodies, is already approved for the treatment of primary and familiar hyperlipidemia and for the treatment of hypercholesterolemia in statin-resistant patients [89,90,91,92,93]. Active and passive immunological approaches against PCSK9, such as vaccines, are now under preclinical evaluation also for cancer treatment [94,95,96,97,98].

As a cancer vaccine delivery system, virus-like particle (VLP)-based vaccines demonstrated to have great immunogenic potential due to their high-density epitope display and their ability to emulate native bacteriophages and viruses in their ability to trigger humoral and cellular immune responses [99,100,101,102], even in preclinical settings of tumor prevention and therapy of several cancer types [102,103,104], including HER2-positive breast cancer [42, 105,106,107,108].

Thus, we sought to investigate whether the combination of two cVLP-based vaccines against HER2 and PCSK9, which separately demonstrated to be effective in triggering neutralizing immune responses [42, 94, 105], could enhance tumor eradication and prolong survival in a preclinical model of HER2-positive breast cancer.

Methods

Vaccine formulation

The system employs a Tag/Catcher conjugation system: AP205 phage capsid cVLP is conjugated with Catcher-HER2ECD (human) or Tag-PCSK9 (mouse). Design, expression and purification of cVLP-HER2 and cVLP-PCSK9 vaccines were performed as previously reported [42, 94].

Cell lines

MamBo89HER2stable cell line was established from a spontaneous mammary carcinoma of a transgenic human HER2 female mouse (FVBhHER2) bred in our animal facility (breeders were originally received from Genentech, South San Francisco, CA, USA) [30]. Mambo89HER2stable, which expresses surface human HER2 at levels comparable to those of 3 + human breast cancer cell lines like BT-474, represents a good preclinical model to test immunological therapies directed against HER2 in immunocompetent mice [30, 42]. MamBo89HER2stable cell line was cultured in DMEM (Thermo Fisher Scientific, Monza, Italy) supplemented with 20% of fetal bovine serum (FBS), 100 U/mL penicillin and 10 μg/mL streptomycin (Thermo Fisher Scientific, Monza, Italy), 30 μg/ml bovine pituitary extract (Corning Life Sciences, USA) and 0.5% v/v MITO Serum Extender (Corning Life Sciences, USA) [30].

Human HER2-positive breast cancer cell line BT-474 (HER2 +  + +) and its trastuzumab-resistant clone C5 (from ATCC) were routinely cultured in RPMI (Thermo Fisher Scientific, Monza, Italy) supplemented with 10% FBS, 100 U/mL penicillin and 10 μg/mL streptomycin. BT-474 cell line (from ATCC) was kindly provided by Dr. S. Pupa (Istituto Nazionale dei Tumori, Milan, Italy);

All cell lines were cultured at 37 °C in a humidified 5% CO2 atmosphere and were split once or twice a week according to density, using 0.05% trypsin–EDTA (Thermo Fisher Scientific, Monza, Italy).

Mice, tumor cells challenge, vaccination and bleedings

FVB female mice were purchased from Charles River (Calco, Lecco, Italy).

18-week-old virgin FVB female mice were challenged with 5 × 106 MamBo89HER2stable cells in the mammary fat pad (m.f.p) of the fourth mammary area.

Vaccines were formulated with AddaVax (Invitrogen, Thermo Fisher Scientific, Waltham, USA), as adjuvant 1:1. The vehicle group (also referred as ‘untreated’) received PBS alone.

The standard dose of vaccine was 10 μg and 5 μg per mouse per administration for cVLP-HER2 vaccine and cVLP-PCSK9 respectively, as reported in previous works [42, 94]. Vaccines were administered intramuscularly (i.m.) in the left hind leg. Vaccinations started two weeks after cell challenge and mice received 6 bi-weekly vaccine administrations.

Mice were bled before each vaccination and two weeks after the last vaccination. Sera were stored at -80 °C and tested through immunological assays.

Tumors were measured with calipers twice a week; tumor volume was calculated as π/6*(√ab)3 where a = maximal tumor diameter and b = maximal tumor diameter perpendicular to a. Mice were euthanized if they showed any signs of distress or when tumor volume reached 2 cm3.

All mice were monitored daily and weighed twice weekly.

All in vivo experiments were performed according to Italian and European laws and were authorized by the Italian Ministry of Health (letter 714–2017-PR).

Enzyme-linked immunosorbent assay (ELISA)

Serum anti-human HER2 IgG levels were quantitatively measured by ELISA immunoassay.

Immunoplate Nunc Maxisorp 96-well microplates (Merck, Darmstadt, Germany) were coated overnight with the HER2ECD or with PCSK9 protein at 1 μg/mL in carbonate bicarbonate buffer (Merck, Darmstadt, Germany) [42, 94].

A standard curve (from 0.04 to 30 ng/mL) with mouse monoclonal antibody 4D5 against human HER2 (Genentech, Roche, San Francisco, USA) was run in parallel.

The following horseradish peroxidase (HRP)-labeled goat anti-mouse Ig antibodies, all from Thermo Fisher Scientific, were used for the detection of: total IgG (1:30,000 dilution), IgM (1:10,000 dilution), IgG1 (1:10,000 dilution), IgG2a (1:5000 dilution), IgG2b (1:10,000 dilution), IgG3 (1:5000 dilution).

PCSK9 protein level in mouse sera was evaluated by Mouse Proprotein Convertase 9/PCSK9 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.

Optical density (OD) was determined with a microplate reader (Sunrise, Tecan, Switzerland).

3D-agar colony growth inhibition

In vitro sensitivity of human breast cancer BT-474 (HER2 +  +  + , trastuzumab-sensitive) and BT-474 C5 (HER2 +  +  + , trastuzumab-resistant) cells to cVLP-HER2 and cVLP-PCSK9-induced antibodies were evaluated in a 3D-agar colony growth assay.

As described in detail previously [109], cells were seeded at 500 cells/well in 24-well plates (Corning Life Sciences, USA) in RPMI supplemented with 10% FBS + 0.33% agar (Lonza Bioscience Solutions, Siena, Italy) with mouse sera diluted 1:100 or trastuzumab (kindly provided by Genentech) diluted at the same concentration as anti-HER2 antibodies in vaccinated mouse sera.

Colonies (diameter > 90 μm) were counted under an inverted microscope equipped with an ocular micrometer in dark-field, 14–30 days after seeding.

Statistical analysis

All statistical analyses were performed using GraphPad Prism (version 9.2.0) (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com).

The Kaplan–Meier tumor-free survival curve, as a method for estimating the survival function from time datapoints, was applied to events of progression (tumor positivity) after an initial tumor regression. Events occurred when no regression was achieved or when a specific tumor volume (≥ 0.01 cm3) was reached after an initial regression. The comparison of the Kaplan–Meier curves was carried out with Log-Rank (Mantel-Cox) test, for detecting differences between groups when the risk of an event is not equally distributed.

Anti-PCSK9 antibodies, serum PCSK9 protein levels and their percentage of inhibition were compared with the unequal variance t-test, also called Welch’s t-test (unpaired). The unpaired Welch’s t-test compares the means of two unmatched groups (e.g. vehicle and cVLPs, in our case), without the assumption that their standard deviations are similar.

The number of colonies in the 3D agar colony inhibition assay was compared with Student’s t-test (unpaired).

Results

cVLP-HER2 and cVLP-PCSK9 vaccines as therapeutic strategies of HER2-positive mammary carcinoma

The therapeutic activity of the combination of cVLP-HER2 and cVLP-PCSK9 vaccines was evaluated against mammary carcinomas induced by the injection of MamBo89HER2stable cells in the mammary fat pad of syngeneic FVB female mice, and compared to the control groups treated with cVLP-HER2 and cVLP-PCSK9 alone or untreated.

Vaccines, formulated with AddaVax adjuvant in a 1:1 ratio, were administered bi-weekly starting 2 weeks after cell challenge, for a total of 6 immunizations (Fig. 1A).

Fig. 1
figure 1

Therapeutic efficacy of cVLP-PCSK9 and cVLP-HER2 vaccines as standalone or combined approach. A Experimental design; B Kaplan–Meier tumor free survival plot, n = 6; *p < 0.05; ***p < 0.01; long-rank test; C Tumor growth curves, each point represents the mean (and SEM) of all mice in each group, n = 6; D Single mice growth curves of each treated group

Full size image

Untreated mice and those treated with the cVLP-PCSK9 vaccine alone developed progressive tumors within 1–2 months after cell injection, whereas 83% of mice vaccinated with cVLP-HER2 and 100% of cVLP-HER2 + cVLP-PCSK9 showed tumor regression from the 40th day after cell challenge (Fig. 1B-D). While both cVLP-HER2 and cVLP-HER2 + cVLP-PCSK9 approaches significantly increased mice survival if compared to untreated and cVLP-PCSK9 treated group, all mice treated with the vaccine combination remained tumor-free even 4 months later, whereas 60% of cVLP-HER2 vaccinated mice experienced tumor relapse (Fig. 1B). Thus, the addition of cVLP-PCSK9 vaccine significantly increased the long-term therapeutic efficacy of cVLP-HER2 (Fig. 1B).

Anti-HER2 antibody response

In FVB mice, cVLP-HER2 + cVLP-PCSK9 vaccination elicited strong (1–2 mg/mL) anti-human HER2 antibody responses (Fig. 2A), comprising different mouse IgG isotypes subclasses, comparable to the ones elicited by cVLP-HER2 vaccination alone. In particular, the polyclonal anti-HER2 antibody response included the IgG subclasses with the strongest antitumoral effect (i.e. IgG2a, IgG2b and IgG3) (Fig. 2B) [110, 111]. The prevalent IgG isotype was IgG1, closely followed by IgG2a and IgG2b, in both the combination therapy and cVLP-HER2 treated groups (Fig. 2B).

Fig. 2
figure 2

Anti-HER2 polyclonal antibody response elicited by cVLP-HER2 vaccine. A Anti-HER2 antibody titers measured by ELISA on human HER2ECD, n = 6. Each point represents the mean (and SEM) of mouse groups shown in Fig. 1; B HER2 specific IgM and IgG isotypes elicited by cVLP-HER2 vaccinations, n = 2–4. Each point represents the mean (and SEM). Histogram reports the mean (and SEM) peak value of each isotype after the last vaccination

Full size image

Anti-PCSK9 antibody response and PCSK9 protein modulation in mouse sera

The binding activity of anti-PCSK9 antibodies to full-length PCSK9 was investigated through ELISA assay.

While anti-PCSK9 antibody levels could barely be detected in the vehicle and cVLP-HER2 treated groups, cVLP-PCSK9 vaccine alone elicited a 137-fold increase in anti-PCSK9 antibody titers, compared to the control group. The anti-PCSK9 response reached a smaller but still significant increase (sevenfold) compared to vehicle treated mice (Fig. 3A, B).

Fig. 3
figure 3

Anti-PCSK9 responses induced by cVLP-PCSK9 vaccine. A Mean (and SEM) of anti-PCSK9 antibody titers measured by ELISA represented as logarithm of Optical Density (OD) value multiplied by dilution factor of the serum sample, n = 6. Vehicle vs. cVLP-PCSK9, at least p < 0.01, from day 56; Vehicle vs. cVLP-HER2 + cVLP-PCSK9, p < 0.05, from day 98; cVLP-HER2 vs. cVLP-PCSK9, at least p < 0.05, from day 56; cVLP-PCSK9 vs. cVLP-HER2 + cVLP-PCSK9, at least p < 0.05, from day 98; cVLP-HER2 vs. cVLP-HER2 + cVLP-PCSK9, p < 0.05, day 98. All statistics were carried out by Welch t-test. B Percentage of the increase of anti-PCSK9 total antibodies after the last vaccination. Histogram reports mean (and SEM) of each group. *p < 0.05 vs Vehicle, + p < 0.05 vs cVLP-HER2, #p < 0.05 at least vs cVLP-HER2 + cVLP-PCSK9 by Welch’s t-test; C PCSK9 protein levels in mouse serum (µg/mL), each point represents the mean (and SEM) for each experimental group, n = 5–6. D Percentage of the increase of anti-PCSK9 protein inhibition after the last vaccination. Histogram reports mean (and SEM) of each group. + p < 0.05 vs cVLP-HER2, #p < 0.05 at least vs cVLP-HER2 + cVLP-PCSK9 by Welch’s t-test

Full size image

The biological effect of the vaccine-induced antibodies was investigated by measuring PCSK9 protein levels in mice sera, resulting in an overall negligible inhibition in the vehicle group and cVLP-HER2 group (Fig. 3C, D). According to PCSK9 antibodies levels, PCSK9 protein levels were significantly reduced in the cVLP-PCSK9 treatment group, while the combination of cVLP-PCSK9 with cVLP-HER2 was associated with an overall less marked inhibition and opsonization of PCSK9; in this group there was a more heterogeneous protein inhibition, as two-thirds of mice presented a decrease in PCSK9 protein levels (data not shown).

Inhibition of human HER2-positive breast cancer cells growth in 3D cultures

To further evaluate the functional activity of the antibodies induced by the vaccines, their inhibitory effect was tested in a 3D-soft agar colony formation assay on human HER2 +  +  + trastuzumab-sensitive, BT-474, and on its trastuzumab-resistant clone BT-474 C5.

Mouse serum from mice immunized with vehicle, cVLP-HER2, cVLP-PCSK9 and cVLP-HER2 combined with cVLP-PCSK9 vaccines were diluted in soft-agar layers. As technical controls, untreated tumor-free mouse sera and trastuzumab were diluted in a similar concentration as the immune sera in the soft-agar layers.

As expected, the BT-474 cell line was significantly inhibited by trastuzumab, whereas the BT474 C5 clone was not inhibited (Fig. 4).

Fig. 4
figure 4

Inhibition of human breast cancer cell 3D agar colony growth by antibodies elicited by vaccinations. Upper panel, colony inhibition assay on BT-474 (HER2 +  +  + , trastuzumab sensitive) and BT-474 C5 (HER2 +  +  + , trastuzumab resistant), n = 2. Each bar represents the mean (and SEM) number of colonies larger than 90 µm as counted in two independent cultures with the aid of a micrometer. *p < 0.05 vs untreated, °p < 0.05 vs cVLP-PCSK9, + p < 0.05 vs cVLP-HER2, by t student’s test, unpaired. Lower panel, representative pictures of live agar colonies were acquired with an inverted microscope (dark-field, 25X)

Full size image

The anti-human HER2 antibodies elicited by cVLP-HER2 + cVLP-PCSK9 and cVLP-HER2 alone significantly impaired 3D-colony formation of both BT-474 and BT-474 C5 cells. Moreover, antibodies elicited by the combined vaccination were more effective than those elicited by the cVLP-HER2 vaccine alone in the inhibition of BT-474 C5 (Fig. 4).

Discussion

In this work, we combined a cVLP-based vaccine against human HER2, which previously demonstrated strong immunogenicity and anti-tumoral activities in preclinical mouse model of HER2-positive breast cancer [42, 105], with a second cVLP-based vaccine directed against mouse PCSK9, developed to manage cholesterol-related disease [94].

Combining cVLP-PCSK9 and cVLP-HER2 vaccines resulted in more efficient and rapid tumor regression and in a significantly higher tumor-free survival, compared to cVLP-HER2 vaccine alone.

In our previous studies [105], we achieved a tumor-free survival of about two years by treating mice with the cVLP-HER2 vaccine combined with Montanide ISA51 as adjuvant. In the present study, we aimed to assess the potential adjuvant effect of the cVLP-PCSK9 vaccine by testing the cVLP-HER2 vaccine under suboptimal conditions. Specifically, we challenged mice with a different cell line and injection dose, using MamBo89HER2stable cells instead of D16_BO_QD cells. Based on our previous findings, AddaVax elicited antibody titers that were five times lower than those achieved with Montanide ISA51 when combined with the cVLP-HER2 vaccine (data not shown), thus we administered the cVLP-HER2 vaccine with AddaVax, in order to not reach the cVLP-HER2 maximal therapeutic efficacy and to shed light on the possible adjuvant effect exerted by the cVLP-PCSK9 vaccine.

The combination of cVLP-HER2 and cVLP-PCSK9 induced strong anti-human HER2 antibody response, slightly higher than those induced by cVLP-HER2 alone, while anti-PCSK9 antibodies levels were reduced in the combined immunization approach if compared to the single treatment with cVLP-PCSK9. Overall, cVLP-PCSK9 alone elicited a humoral response that inhibited PCSK9 protein levels and, less prominently, in combination with cVLP-HER2. The lower, but still significant compared to controls, anti-PCSK9 response observed in mice receiving the cVLP-PCSK9 combined with cVLP-HER2 could be linked to the immunodominance of HER2 or to potential vaccine interferences [112]. To further enhance cVLP-PCSK9 antibody response when administered with cVLP-HER2, different immunization schedules will be evaluated.

The main aim of the present study was to investigate the therapeutic activity of cVLP-HER2 + cVLP-PCSK9 against HER2-positive mammary carcinomas, but we also had the opportunity to preliminarily evaluate its safety in mice. Weight curves of unvaccinated and vaccinated mice overlapped (Supplementary Figures S1 and S2). As judged from the constant monitoring of the mice, vaccines either alone or combined did not compromise the general health status of mice. Moreover, no signs of suffering or distress were reached according to the humane endpoints. At necropsy, liver and kidney tissues were not altered and macroscopically no signs of local toxicity (e.g. redness, swelling or accumulation of suspension in the hind leg) were reported.

To better evaluate the functional activity of antibodies induced by the vaccines, their inhibitory activity was investigated on human HER2 +  +  + breast cancer cells BT-474 (trastuzumab-sensitive) and on its trastuzumab-resistant clone BT-474 C5, in three-dimensional colonies in soft-agar. As expected, anti-HER2 antibodies elicited by cVLP-HER2 + cVLP-PCSK9 vaccines exerted great inhibitory abilities on the HER2-positive trastuzumab-sensitive cell line, similar to the ones elicited by cVLP-HER2 alone. Interestingly, on the trastuzumab-resistant cell line, antibodies elicited by the combination therapy showed a higher inhibitory effect than cVLP-HER2 alone antibodies, suggesting that this therapeutic regimen might be more effective in managing heterogeneous human HER2-positive tumors in which trastuzumab-resistant and trastuzumab-sensitive subclones coexist.

Mechanistically, the higher therapeutic effect of the cVLP-HER2 and cVLP-PCSK9 combined immunization protocol was not strictly related to the inhibition of PCSK9, as more information about the anti-tumor activity of the inhibition of PCSK9 in our preclinical model need to be collected. We hypothesize that the great in vivo inhibition showed by this therapeutical approach is strictly related to the anti-human HER2 antibodies induced both by the combined approach and by cVLP-HER2 alone. In our previous results [113], we saw that the inhibition of HER2/neu carcinogenesis depends on antibody production which can be related to B cells and IFN-γ release. Thus, we may expect that T cell depletion results in a reduced IFN-γ release and consequently in a lower antibody production which can impair vaccine therapeutic efficacy. While not reaching statistical significance, antibodies induced by the combined vaccination approach are higher than those induced by cVLP-HER2 alone, contributing to the stronger anti-tumoral effect together with an increase in antibodies’ affinity and avidity. The in vitro experiments on human cell lines confirmed that an immune mechanism by which anti-HER2 antibodies elicited by vaccines exert their anti-tumor activity is through direct inhibition of HER2-positive tumor cells.

Moreover, the induction of a polyclonal antibody response against HER2, involving in particular the IgG2a subtype, could indicate the activation of a T helper type 1 (Th1) immune response, able to enhance tumor antigens presentation, activation of cytotoxic T lymphocytes or to directly kill tumor cells by death-receptor activation by cytokines on cancer cells [114,115,116].

In addition, in HER2 overexpressing tumors, where the signaling receptor is colocalized with cholesterol-rich signaling niches called lipid rafts, the role of cholesterol and PCSK9 is not only on a metabolic level, but also on a signaling level, as these lipid structures can affect HER2 stability on the cell membrane and its internalization after anti-HER2 targeted treatments [28, 40, 117].

Another potential mechanism behind the increased antitumoral effect exerted by the combination of the two vaccines could be the modulation of low-density lipoprotein receptor (LDLR). Partial inhibition of PCSK9 may have lifted the blockage on LDLR on lymphocytes, leading to enhanced TCR recycling and activation of CD8 + effector T cells [118]. This effect is independent of cholesterol levels, but depends on PCSK9 biological functions [118]. Therefore, it is crucial to gain a broader understanding of the PCSK9 protein level threshold associated with its functionality.

Indeed, the enhanced antitumoral effect of the combination therapy could be associated to an impairment of MHC-I degradation, which was reported to be caused by binding with PCSK9 [119, 120]. Thus, the inhibition of PCSK9 could have enhanced antigen-presenting abilities and activated cell-mediated immune responses. Deeper analyses are necessary to understand whether the effect on tumor growth is attributed to the effects of PCSK9 on the immune system.

In summary, our results suggest that anti-PCSK9 treatments can enhance anti-HER2 therapies efficacy, also against trastuzumab-resistant cells, confirming that VLP-based vaccines are a valid therapeutic strategy able to induce strong humoral responses and anti-tumor effects.

Conclusions

HER2-targeted therapies have revolutionized the treatment of HER2-positive breast cancer patients; however, resistance and tumor relapse remain considerable challenges.

Our results showed that cVLP-PCSK9 vaccine had adjuvant activity when combined with cVLP-HER2 vaccine, enhancing its therapeutic efficacy against HER2-positive breast cancer and mice tumor-free survival.

On a trastuzumab-resistant cell line, the antibodies elicited by the combined therapy showed a higher inhibitory effect than ones elicited by cVLP-HER2 alone, holding promise in overcoming the challenges posed by resistance and incomplete responses to HER2-targeted therapy.

Availability of data and materials

Not applicable.

Abbreviations

ADC:

Antibody–drug conjugate

cVLP:

Capsid virus-like particle

ECD:

Extracellular domain

FA:

Fatty acid

FBS:

Fetal bovine serum

GOF:

Gain of function

HER2:

Human epidermal growth factor receptor 2

LDLR:

Low-density lipoprotein receptor

mAb:

Monoclonal antibody

MHC:

Major histocompatibility complex

OD:

Optical density

PCSK9:

Proprotein convertase subtilisin/kexin type 9

TCR:

T-cell receptor

Th1:

T-helper type 1

TKIs:

Tyrosin-kinase inhibitors

References

  1. Testa U, Castelli G, Pelosi E. breast cancer: a molecularly heterogenous disease needing subtype-specific treatments. Med Sci. 2020;8:18. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/medsci8010018.

    Article  CAS  Google Scholar 

  2. Guo L, Kong D, Liu J, Zhan L, Luo L, Zheng W, Zheng Q, Chen C, Sun S. Breast cancer heterogeneity and its implication in personalized precision therapy. Exp Hematol Oncol. 2023;12:3. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40164-022-00363-1.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Lüönd F, Tiede S, Christofori G. Breast cancer as an example of tumour heterogeneity and tumour cell plasticity during malignant progression. Br J Cancer. 2021;125:164–75. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41416-021-01328-7.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Valenza C, Guidi L, Battaiotto E, Trapani D, Sartore Bianchi A, Siena S, Curigliano G. Targeting HER2 heterogeneity in breast and gastrointestinal cancers. Trends Cancer. 2024;10:113–23. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.trecan.2023.11.001.

    Article  CAS  PubMed  Google Scholar 

  5. Xu Q, Xu X, Tang H, Yan J, Li J, Bao H, Wu X, Shao Y, Luo C, Wen H, Jin J, Ying J. Exploring potential molecular resistance and clonal evolution in advanced HER2-positive gastric cancer under trastuzumab therapy. Oncogenesis. 2023;12:21. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41389-023-00466-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Laé M, Couturier J, Oudard S, Radvanyi F, Beuzeboc P, Vieillefond A. Assessing HER2 gene amplification as a potential target for therapy in invasive urothelial bladder cancer with a standardized methodology: results in 1005 patients. Ann Oncol. 2010;21:815–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/annonc/mdp488.

    Article  PubMed  Google Scholar 

  7. S. Ahcene Djaballah, F. Daniel, A. Milani, G. Ricagno, S. Lonardi, HER2 in colorectal cancer: the long and winding road from negative predictive factor to positive actionable target, American Society of Clinical Oncology Educational Book (2022) 219–232. https://doiorg.publicaciones.saludcastillayleon.es/10.1200/EDBK_351354.

  8. Ménard S, Pupa SM, Campiglio M, Tagliabue E. Biologic and therapeutic role of HER2 in cancer. Oncogene. 2003;22:6570–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/sj.onc.1206779.

    Article  CAS  PubMed  Google Scholar 

  9. Eliyatkin N, Yalcin E, Zengel B, Aktaş S, Vardar E. Molecular classification of breast carcinoma: from traditional, old-fashioned way to a new age, and a new way. J Breasth Health. 2015;11:59–66. https://doiorg.publicaciones.saludcastillayleon.es/10.5152/tjbh.2015.1669.

    Article  Google Scholar 

  10. Guidi L, Pellizzari G, Tarantino P, Valenza C, Curigliano G. Resistance to antibody-drug conjugates targeting HER2 in breast cancer: molecular landscape and future challenges. Cancers. 2023;15:1130. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cancers15041130.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Choong GM, Cullen GD, O’Sullivan CC. Evolving standards of care and new challenges in the management of HER2-positive breast cancer. CA A Cancer J Clinicians. 2020;70:355–74. https://doiorg.publicaciones.saludcastillayleon.es/10.3322/caac.21634.

    Article  Google Scholar 

  12. M.F. Rimawi, C. De Angelis, R. Schiff, Resistance to Anti-HER2 Therapies in Breast Cancer, American Society of Clinical Oncology Educational Book (2015). https://doiorg.publicaciones.saludcastillayleon.es/10.14694/EdBook_AM.2015.35.e157.

  13. Vernieri C, Milano M, Brambilla M, Mennitto A, Maggi C, Cona MS, Prisciandaro M, Fabbroni C, Celio L, Mariani G, Bianchi GV, Capri G, De Braud F. Resistance mechanisms to anti-HER2 therapies in HER2-positive breast cancer: current knowledge, new research directions and therapeutic perspectives. Crit Rev Oncol Hematol. 2019;139:53–66. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.critrevonc.2019.05.001.

    Article  PubMed  Google Scholar 

  14. Barok M, Joensuu H, Isola J. Trastuzumab emtansine: mechanisms of action and drug resistance. Breast Cancer Res. 2014;16:209. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/bcr3621.

    Article  PubMed  PubMed Central  Google Scholar 

  15. D. De Melo Gagliato, D. Leonardo Fontes Jardim, M.S.P. Marchesi, G.N. Hortobagyi, Mechanisms of resistance and sensitivity to anti-HER2 therapies in HER2+ breast cancer, Oncotarget. 2016; 7: 64431–64446. https://doiorg.publicaciones.saludcastillayleon.es/10.18632/oncotarget.7043.

  16. Wu X, Yang H, Yu X, Qin J-J. Drug-resistant HER2-positive breast cancer: molecular mechanisms and overcoming strategies. Front Pharmacol. 2022;13:1012552. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fphar.2022.1012552.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Swain SM, Shastry M, Hamilton E. Targeting HER2-positive breast cancer: advances and future directions. Nat Rev Drug Discov. 2023;22:101–26. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41573-022-00579-0.

    Article  CAS  PubMed  Google Scholar 

  18. Swain SM, Macharia H, Cortes J, Dang C, Gianni L, Hurvitz SA, Jackisch C, Schneeweiss A, Slamon D, Valagussa P, Du Toit Y, Heinzmann D, Knott A, Song C, Cortazar P. Event-free survival in patients with early HER2-positive breast cancer with a pathological complete response after HER2-targeted therapy: a pooled analysis. Cancers. 2022;14:5051. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cancers14205051.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Saleh K, Khoury R, Khalife N, Chahine C, Ibrahim R, Tikriti Z, Le Cesne A. Mechanisms of action and resistance to anti-HER2 antibody-drug conjugates in breast cancer. Cancer Drug Resist. 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.20517/cdr.2024.06.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Nahta R, Esteva FJ. HER2 therapy: molecular mechanisms of trastuzumab resistance. Breast Cancer Res. 2006;8:215. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/bcr1612.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chandarlapaty S, Sakr RA, Giri D, Patil S, Heguy A, Morrow M, Modi S, Norton L, Rosen N, Hudis C, King TA. Frequent mutational activation of the PI3K-AKT pathway in trastuzumab-resistant breast cancer. Clin Cancer Res. 2012;18:6784–91. https://doiorg.publicaciones.saludcastillayleon.es/10.1158/1078-0432.CCR-12-1785.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lu Y, Zi X, Pollak M. Molecular mechanisms underlying IGF-I-induced attenuation of the growth-inhibitory activity of trastuzumab (Herceptin) on SKBR3 breast cancer cells, Intl Journal of. Cancer. 2004;108:334–41. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/ijc.11445.

    Article  CAS  Google Scholar 

  23. De Giovanni C, Landuzzi L, Palladini A, Ianzano M, Nicoletti G, Ruzzi F, Amici A, Croci S, Nanni P, Lollini P-L. Cancer vaccines Co-targeting HER2/Neu and IGF1R. Cancers. 2019;11:517. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cancers11040517.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pohlmann PR, Mayer IA, Mernaugh R. Resistance to trastuzumab in breast cancer. Clin Cancer Res. 2009;15:7479–91. https://doiorg.publicaciones.saludcastillayleon.es/10.1158/1078-0432.CCR-09-0636.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Castagnoli L, Ghedini GC, Koschorke A, Triulzi T, Dugo M, Gasparini P, Casalini P, Palladini A, Iezzi M, Lamolinara A, Lollini PL, Nanni P, Chiodoni C, Tagliabue E, Pupa SM. Pathobiological implications of the d16HER2 splice variant for stemness and aggressiveness of HER2-positive breast cancer. Oncogene. 2017;36:1721–32. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/onc.2016.338.

    Article  CAS  PubMed  Google Scholar 

  26. Castagnoli L, Ladomery M, Tagliabue E, Pupa SM. The d16HER2 splice variant: a friend or foe of HER2-positive cancers? Cancers. 2019;11:902. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cancers11070902.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sung M, Tan X, Lu B, Golas J, Hosselet C, Wang F, Tylaska L, King L, Zhou D, Dushin R, Myers JS, Rosfjord E, Lucas J, Gerber H-P, Loganzo F. Caveolae-mediated endocytosis as a novel mechanism of resistance to trastuzumab emtansine (T-DM1). Mol Cancer Ther. 2018;17:243–53. https://doiorg.publicaciones.saludcastillayleon.es/10.1158/1535-7163.MCT-17-0403.

    Article  CAS  PubMed  Google Scholar 

  28. Ruzzi F, Cappello C, Semprini MS, Scalambra L, Angelicola S, Pittino OM, Landuzzi L, Palladini A, Nanni P, Lollini P-L. Lipid rafts, caveolae, and epidermal growth factor receptor family: friends or foes? Cell Commun Signal. 2024;22:489. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12964-024-01876-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Giugliano F, Carnevale Schianca A, Corti C, Ivanova M, Bianco N, Dellapasqua S, Criscitiello C, Fusco N, Curigliano G, Munzone E. Unlocking the resistance to Anti-HER2 treatments in breast cancer: the issue of HER2 spatial distribution. Cancers. 2023;15:1385.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Giusti V, Ruzzi F, Landuzzi L, Ianzano ML, Laranga R, Nironi E, Scalambra L, Nicoletti G, De Giovanni C, Olivero M, Arigoni M, Calogero R, Nanni P, Palladini A, Lollini P-L. Evolution of HER2-positive mammary carcinoma: HER2 loss reveals claudin-low traits in cancer progression. Oncogenesis. 2021;10:77. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41389-021-00360-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hamilton E, Shastry M, Shiller SM, Ren R. Targeting HER2 heterogeneity in breast cancer. Cancer Treat Rev. 2021;100: 102286. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ctrv.2021.102286.

    Article  CAS  PubMed  Google Scholar 

  32. Z. Li, O. Metzger Filho, G. Viale, P. dell’Orto, L. Russo, M.-A. Goyette, A. Kamat, D.A. Yardley, V. Gupta Abramson, C.L. Arteaga, L.M. Spring, K. Chiotti, C. Halsey, A.G. Waks, T.A. King, S.C. Lester, J.R. Bellon, E.P. Winer, P.T. Spellman, I.E. Krop, K. Polyak, HER2 heterogeneity and treatment response–associated profiles in HER2-positive breast cancer in the NCT02326974 clinical trial, Journal of Clinical Investigation 134 (2024) e176454. https://doiorg.publicaciones.saludcastillayleon.es/10.1172/JCI176454.

  33. Koene HR, Kleijer M, Algra J, Roos D, von dem Borne AE, de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood. 1997;90:1109–14.

    Article  CAS  PubMed  Google Scholar 

  34. Maadi H, Soheilifar MH, Choi W-S, Moshtaghian A, Wang Z. Trastuzumab mechanism of action; 20 years of research to unravel a dilemma. Cancers. 2021;13:3540. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cancers13143540.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Nami B, Maadi H, Wang Z. Mechanisms underlying the action and synergism of trastuzumab and pertuzumab in targeting HER2-positive breast cancer. Cancers. 2018;10:342. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cancers10100342.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cortés J, Fumoleau P, Bianchi GV, Petrella TM, Gelmon K, Pivot X, Verma S, Albanell J, Conte P, Lluch A, Salvagni S, Servent V, Gianni L, Scaltriti M, Ross GA, Dixon J, Szado T, Baselga J. Pertuzumab monotherapy after trastuzumab-based treatment and subsequent reintroduction of trastuzumab: activity and tolerability in patients with advanced human epidermal growth factor receptor 2–positive breast cancer. JCO. 2012;30:1594–600. https://doiorg.publicaciones.saludcastillayleon.es/10.1200/JCO.2011.37.4207.

    Article  CAS  Google Scholar 

  37. Loi S, Giobbie-Hurder A, Gombos A, Bachelot T, Hui R, Curigliano G, Campone M, Biganzoli L, Bonnefoi H, Jerusalem G, Bartsch R, Rabaglio-Poretti M, Kammler R, Maibach R, Smyth MJ, Di Leo A, Colleoni M, Viale G, Regan MM, André F, Fumagalli D, Gelber RD, Goulioti T, Hiltbrunner A, Hui R, Roschitzki H, Ruepp B, Boyle F, Stahel R, Aebi S, Coates AS, Goldhirsch A, Karlsson P, Kössler I, Fournarakou S, Gasca A, Pfister R, Ribeli-Hofmann S, Weber M, Celotto D, Comune C, Frapolli M, Sánchez-Hohl M, Huang H, Mahoney C, Price K, Scott K, Shaw H, Fischer S, Greco M, King C, Andrighetto S, Piccart-Gebhart M, Findlay H, Jenkins M, Karantza V, Mejia J, Schneier P. Pembrolizumab plus trastuzumab in trastuzumab-resistant, advanced, HER2-positive breast cancer (PANACEA): a single-arm, multicentre, phase 1b–2 trial. Lancet Oncol. 2019;20:371–82. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S1470-2045(18)30812-X.

    Article  CAS  PubMed  Google Scholar 

  38. Hamilton E, Shapiro CL, Petrylak D, Boni V, Martin M, Conte GD, Cortes J, Agarwal L, Arkenau H-T, Tan AR, Debruyne P, Minchom A, Rutten A, Valdes-Albini F, Yu EY, Augustine B, D’Amelio A, Barrios D, Hurvitz SA. Abstract PD3–07: trastuzumab deruxtecan (T-DXd; DS-8201) with nivolumab in patients with HER2-expressing, advanced breast cancer: a 2-part, phase 1b, multicenter, open-label study. Cancer Res. 2021. https://doiorg.publicaciones.saludcastillayleon.es/10.1158/1538-7445.SABCS20-PD3-07.

    Article  Google Scholar 

  39. Emens LA, Esteva FJ, Beresford M, Saura C, De Laurentiis M, Kim S-B, Im S-A, Wang Y, Salgado R, Mani A, Shah J, Lambertini C, Liu H, De Haas SL, Patre M, Loi S. Trastuzumab emtansine plus atezolizumab versus trastuzumab emtansine plus placebo in previously treated, HER2-positive advanced breast cancer (KATE2): a phase 2, multicentre, randomised, double-blind trial. Lancet Oncol. 2020;21:1283–95. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S1470-2045(20)30465-4.

    Article  CAS  PubMed  Google Scholar 

  40. Pereira PMR, Mandleywala K, Monette S, Lumish M, Tully KM, Panikar SS, Cornejo M, Mauguen A, Ragupathi A, Keltee NC, Mattar M, Janjigian YY, Lewis JS. Caveolin-1 temporal modulation enhances antibody drug efficacy in heterogeneous gastric cancer. Nat Commun. 2022;13:2526. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41467-022-30142-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Heater NK, Warrior S, Lu J. Current and future immunotherapy for breast cancer. J Hematol Oncol. 2024;17:131. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13045-024-01649-z.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Palladini A, Thrane S, Janitzek CM, Pihl J, Clemmensen SB, De Jongh WA, Clausen TM, Nicoletti G, Landuzzi L, Penichet ML, Balboni T, Ianzano ML, Giusti V, Theander TG, Nielsen MA, Salanti A, Lollini P-L, Nanni P, Sander AF. Virus-like particle display of HER2 induces potent anti-cancer responses. OncoImmunology. 2018;7: e1408749. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/2162402X.2017.1408749.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Clay TM, Osada T, Hartman ZC, Hobeika A, Devi G, Morse MA, Kim Lyerly H. Polyclonal immune responses to antigens associated with cancer signaling pathways and new strategies to enhance cancer vaccines. Immunol Res. 2011;49:235–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Triulzi C, Vertuani S, Curcio C, Antognoli A, Seibt J, Akusjärvi G, Wei W-Z, Cavallo F, Kiessling R. Antibody-dependent natural killer cell-mediated cytotoxicity engendered by a kinase-inactive human HER2 adenovirus-based vaccination mediates resistance to breast tumors. Can Res. 2010;70:7431–41. https://doiorg.publicaciones.saludcastillayleon.es/10.1158/0008-5472.CAN-10-0493.

    Article  CAS  Google Scholar 

  45. Liu J, Fu M, Wang M, Wan D, Wei Y, Wei X. Cancer vaccines as promising immuno-therapeutics: platforms and current progress. J Hematol Oncol. 2022;15:28. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13045-022-01247-x.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Paston SJ, Brentville VA, Symonds P, Durrant LG. Cancer vaccines, adjuvants, and delivery systems. Front Immunol. 2021;12: 627932. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fimmu.2021.627932.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lollini P-L, Nicoletti G, Landuzzi L, De Giovanni C, Nanni P. Immunoprevention and immunotherapy of mammary carcinoma: immunoprevention of cancer. Breast J. 2010;16:S39–41. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1524-4741.2010.01002.x.

    Article  PubMed  Google Scholar 

  48. Lollini P-L, Cavallo F, Nanni P, Forni G. Vaccines for tumour prevention. Nat Rev Cancer. 2006;6:204–16. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nrc1815.

    Article  CAS  PubMed  Google Scholar 

  49. Nanni P, Nicoletti G, Palladini A, Croci S, Murgo A, Antognoli A, Landuzzi L, Fabbi M, Ferrini S, Musiani P, Iezzi M, De Giovanni C, Lollini P-L. Antimetastatic activity of a preventive cancer vaccine. Can Res. 2007;67:11037–44. https://doiorg.publicaciones.saludcastillayleon.es/10.1158/0008-5472.CAN-07-2499.

    Article  CAS  Google Scholar 

  50. Hanahan D. Hallmarks of cancer: new dimensions. Cancer Disc. 2022;12:31–46. https://doiorg.publicaciones.saludcastillayleon.es/10.1158/2159-8290.CD-21-1059.

    Article  CAS  Google Scholar 

  51. Mullen PJ, Yu R, Longo J, Archer MC, Penn LZ. The interplay between cell signalling and the mevalonate pathway in cancer. Nat Rev Cancer. 2016;16:718–31. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nrc.2016.76.

    Article  CAS  PubMed  Google Scholar 

  52. Cai D, Wang J, Gao B, Li J, Wu F, Zou JX, Xu J, Jiang Y, Zou H, Huang Z, Borowsky AD, Bold RJ, Lara PN, Li JJ, Chen X, Lam KS, To K-F, Kung H-J, Fiehn O, Zhao R, Evans RM, Chen H-W. RORγ is a targetable master regulator of cholesterol biosynthesis in a cancer subtype. Nat Commun. 2019;10:4621. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41467-019-12529-3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Yue S, Li J, Lee S-Y, Lee HJ, Shao T, Song B, Cheng L, Masterson TA, Liu X, Ratliff TL, Cheng J-X. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. Cell Metab. 2014;19:393–406. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.cmet.2014.01.019.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Mayengbam SS, Singh A, Pillai AD, Bhat MK. Influence of cholesterol on cancer progression and therapy. Transl Oncol. 2021;14: 101043. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.tranon.2021.101043.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer’s achilles’ heel. Cancer Cell. 2008;13:472–82. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ccr.2008.05.005.

    Article  CAS  PubMed  Google Scholar 

  56. Yoon S, Lee M-Y, Park SW, Moon J-S, Koh Y-K, Ahn Y-H, Park B-W, Kim K-S. Up-regulation of acetyl-CoA carboxylase α and fatty acid synthase by human epidermal growth factor receptor 2 at the translational level in breast cancer cells. J Biol Chem. 2007;282:26122–31. https://doiorg.publicaciones.saludcastillayleon.es/10.1074/jbc.M702854200.

    Article  CAS  PubMed  Google Scholar 

  57. Ligorio F, Pellegrini I, Castagnoli L, Vingiani A, Lobefaro R, Zattarin E, Santamaria M, Pupa SM, Pruneri G, De Braud F, Vernieri C. Targeting lipid metabolism is an emerging strategy to enhance the efficacy of anti-HER2 therapies in HER2-positive breast cancer. Cancer Lett. 2021;511:77–87. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.canlet.2021.04.023.

    Article  CAS  PubMed  Google Scholar 

  58. Ligorio F, Di Cosimo S, Verderio P, Ciniselli CM, Pizzamiglio S, Castagnoli L, Dugo M, Galbardi B, Salgado R, Loi S, Michiels S, Triulzi T, Tagliabue E, El-Abed S, Izquierdo M, De Azambuja E, Nuciforo P, Huober J, Moscetti L, Janni W, Coccia-Portugal MA, Corsetto PA, Belfiore A, Lorenzini D, Daidone MG, Vingiani A, Gianni L, Pupa SM, Bianchini G, Pruneri G, Vernieri C. Predictive role of CD36 expression in HER2-positive breast cancer patients receiving neoadjuvant trastuzumab. JNCI J Natl Cancer Ins. 2022;114:1720–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/jnci/djac126.

    Article  CAS  Google Scholar 

  59. Castagnoli L, Corso S, Franceschini A, Raimondi A, Bellomo SE, Dugo M, Morano F, Prisciandaro M, Brich S, Belfiore A, Vingiani A, Di Bartolomeo M, Pruneri G, Tagliabue E, Giordano S, Pietrantonio F, Pupa SM. Fatty acid synthase as a new therapeutic target for HER2-positive gastric cancer. Cell Oncol. 2023;46:661–76. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s13402-023-00769-x.

    Article  CAS  Google Scholar 

  60. Pascual G, Avgustinova A, Mejetta S, Martín M, Castellanos A, Attolini CS-O, Berenguer A, Prats N, Toll A, Hueto JA, Bescós C, Di Croce L, Benitah SA. Targeting metastasis-initiating cells through the fatty acid receptor CD36. Nature. 2017;541:41–5. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nature20791.

    Article  CAS  PubMed  Google Scholar 

  61. Kuhajda FP, Jenner K, Wood FD, Hennigar RA, Jacobs LB, Dick JD, Pasternack GR. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci USA. 1994;91:6379–83. https://doiorg.publicaciones.saludcastillayleon.es/10.1073/pnas.91.14.6379.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Menendez JA, Vellon L, Lupu R. Antitumoral actions of the anti-obesity drug orlistat (Xenical™) in breast cancer cells: blockade of cell cycle progression, promotion of apoptotic cell death and PEA3-mediated transcriptional repression of Her2/neu (erbB-2) oncogene. Ann Oncol. 2005;16:1253–67. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/annonc/mdi239.

    Article  CAS  PubMed  Google Scholar 

  63. Murai T. Cholesterol lowering: role in cancer prevention and treatment. Biol Chem. 2015;396:1–11. https://doiorg.publicaciones.saludcastillayleon.es/10.1515/hsz-2014-0194.

    Article  CAS  PubMed  Google Scholar 

  64. De Gonzalo-Calvo D, López-Vilaró L, Nasarre L, Perez-Olabarria M, Vázquez T, Escuin D, Badimon L, Barnadas A, Lerma E, Llorente-Cortés V. Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential: a molecular and clinicopathological study. BMC Cancer. 2015;15:460. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-015-1469-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Villa GR, Hulce JJ, Zanca C, Bi J, Ikegami S, Cahill GL, Gu Y, Lum KM, Masui K, Yang H, Rong X, Hong C, Turner KM, Liu F, Hon GC, Jenkins D, Martini M, Armando AM, Quehenberger O, Cloughesy TF, Furnari FB, Cavenee WK, Tontonoz P, Gahman TC, Shiau AK, Cravatt BF, Mischel PS. An LXR-cholesterol axis creates a metabolic co-dependency for brain cancers. Cancer Cell. 2016;30:683–93. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ccell.2016.09.008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Voisin M, De Medina P, Mallinger A, Dalenc F, Huc-Claustre E, Leignadier J, Serhan N, Soules R, Ségala G, Mougel A, Noguer E, Mhamdi L, Bacquié E, Iuliano L, Zerbinati C, Lacroix-Triki M, Chaltiel L, Filleron T, Cavaillès V, Al Saati T, Rochaix P, Duprez-Paumier R, Franchet C, Ligat L, Lopez F, Record M, Poirot M, Silvente-Poirot S. Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor. Proc Natl Acad Sci USA. 2017. https://doiorg.publicaciones.saludcastillayleon.es/10.1073/pnas.1707965114.

    Article  PubMed  PubMed Central  Google Scholar 

  67. Chen Y, Hughes-Fulford M. Human prostate cancer cells lack feedback regulation of low-density lipoprotein receptor and its regulator, SREBP2. Int J Cancer. 2001;91:41–5. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/1097-0215(20010101)91:1%3c41::AID-IJC1009%3e3.0.CO;2-2.

    Article  CAS  PubMed  Google Scholar 

  68. Antalis CJ, Arnold T, Rasool T, Lee B, Buhman KK, Siddiqui RA. High ACAT1 expression in estrogen receptor negative basal-like breast cancer cells is associated with LDL-induced proliferation. Breast Cancer Res Treat. 2010;122:661–70. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s10549-009-0594-8.

    Article  CAS  PubMed  Google Scholar 

  69. Wang J, Tan M, Ge J, Zhang P, Zhong J, Tao L, Wang Q, Tong X, Qiu J. Lysosomal acid lipase promotes cholesterol ester metabolism and drives clear cell renal cell carcinoma progression. Cell Prolif. 2018;51: e12452. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/cpr.12452.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Cruz PMR, Mo H, McConathy WJ, Sabnis N, Lacko AG. The role of cholesterol metabolism and cholesterol transport in carcinogenesis: a review of scientific findings, relevant to future cancer therapeutics. Front Pharmacol. 2013;4:119. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fphar.2013.00119.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Kidani Y, Elsaesser H, Hock MB, Vergnes L, Williams KJ, Argus JP, Marbois BN, Komisopoulou E, Wilson EB, Osborne TF, Graeber TG, Reue K, Brooks DG, Bensinger SJ. Sterol regulatory element–binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity. Nat Immunol. 2013;14:489–99. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/ni.2570.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Ma X, Bi E, Lu Y, Su P, Huang C, Liu L, Wang Q, Yang M, Kalady MF, Qian J, Zhang A, Gupte AA, Hamilton DJ, Zheng C, Yi Q. Cholesterol induces CD8+ T cell exhaustion in the tumor microenvironment. Cell Metab. 2019;30:143-156.e5. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.cmet.2019.04.002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Swamy M, Beck-Garcia K, Beck-Garcia E, Hartl FA, Morath A, Yousefi OS, Dopfer EP, Molnár E, Schulze AK, Blanco R, Borroto A, Martín-Blanco N, Alarcon B, Höfer T, Minguet S, Schamel WWA. A cholesterol-based allostery model of T cell receptor phosphorylation. Immunity. 2016;44:1091–101. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.immuni.2016.04.011.

    Article  CAS  PubMed  Google Scholar 

  74. Okoye I, Namdar A, Xu L, Crux N, Elahi S. Atorvastatin downregulates co-inhibitory receptor expression by targeting Ras-activated mTOR signaling. Oncotarget. 2017;8:98215–32.

    Article  PubMed  PubMed Central  Google Scholar 

  75. Yang Y, Li C, Liu T, Dai X, Bazhin AV. Myeloid-derived suppressor cells in tumors: from mechanisms to antigen specificity and microenvironmental regulation. Front Immunol. 2020;11:1371. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fimmu.2020.01371.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Condamine T, Dominguez GA, Youn J-I, Kossenkov AV, Mony S, Alicea-Torres K, Tcyganov E, Hashimoto A, Nefedova Y, Lin C, Partlova S, Garfall A, Vogl DT, Xu X, Knight SC, Malietzis G, Lee GH, Eruslanov E, Albelda SM, Wang X, Mehta JL, Bewtra M, Rustgi A, Hockstein N, Witt R, Masters G, Nam B, Smirnov D, Sepulveda MA, Gabrilovich DI. Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients. Sci Immunol. 2016. https://doiorg.publicaciones.saludcastillayleon.es/10.1126/sciimmunol.aaf8943.

    Article  PubMed  PubMed Central  Google Scholar 

  77. Park J, Lee SE, Hur J, Hong EB, Choi J-I, Yang J-M, Kim J-Y, Kim Y-C, Cho H-J, Peters JM, Ryoo S-B, Kim YT, Kim H-S. M-CSF from cancer cells induces fatty acid synthase and PPARβ/δ activation in tumor myeloid cells, leading to tumor progression. Cell Rep. 2015;10:1614–25. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.celrep.2015.02.024.

    Article  CAS  PubMed  Google Scholar 

  78. King RJ, Singh PK, Mehla K. The cholesterol pathway: impact on immunity and cancer. Trends Immunol. 2022;43:78–92. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.it.2021.11.007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Herber DL, Cao W, Nefedova Y, Novitskiy SV, Nagaraj S, Tyurin VA, Corzo A, Cho H-I, Celis E, Lennox B, Knight SC, Padhya T, McCaffrey TV, McCaffrey JC, Antonia S, Fishman M, Ferris RL, Kagan VE, Gabrilovich DI. Lipid accumulation and dendritic cell dysfunction in cancer. Nat Med. 2010;16:880–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nm.2172.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Cao W, Ramakrishnan R, Tuyrin VA, Veglia F, Condamine T, Amoscato A, Mohammadyani D, Johnson JJ, Min Zhang L, Klein-Seetharaman J, Celis E, Kagan VE, Gabrilovich DI. Oxidized lipids block antigen cross-presentation by dendritic cells in cancer. J Immunol. 2014;192:2920–31. https://doiorg.publicaciones.saludcastillayleon.es/10.4049/jimmunol.1302801.

    Article  CAS  Google Scholar 

  81. Guo Q, Feng X, Zhou Y. PCSK9 variants in familial hypercholesterolemia: a comprehensive synopsis. Front Genet. 2020;11:1020. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fgene.2020.01020.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Mahboobnia K, Pirro M, Marini E, Grignani F, Bezsonov EE, Jamialahmadi T, Sahebkar A. PCSK9 and cancer: rethinking the link. Biomed Pharmacother. 2021;140: 111758. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.biopha.2021.111758.

    Article  CAS  PubMed  Google Scholar 

  83. Seidah NG. The PCSK9 discovery, an inactive protease with varied functions in hypercholesterolemia, viral infections, and cancer. J Lipid Res. 2021;62: 100130. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jlr.2021.100130.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Hsu C-Y, Abdulrahim MN, Mustafa MA, Omar TM, Balto F, Pineda I, Khudair TT, Ubaid M, Ali MS. The multifaceted role of PCSK9 in cancer pathogenesis, tumor immunity, and immunotherapy. Med Oncol. 2024;41:202. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s12032-024-02435-0.

    Article  CAS  PubMed  Google Scholar 

  85. Yang Q-C, Wang S, Liu Y-T, Song A, Wu Z-Z, Wan S-C, Li H-M, Sun Z-J. Targeting PCSK9 reduces cancer cell stemness and enhances antitumor immunity in head and neck cancer. iScience. 2023;26:106916. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.isci.2023.106916.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Wang R, Liu H, He P, An D, Guo X, Zhang X, Feng M. Inhibition of PCSK9 enhances the antitumor effect of PD-1 inhibitor in colorectal cancer by promoting the infiltration of CD8+ T cells and the exclusion of treg cells. Front Immunol. 2022;13: 947756. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fimmu.2022.947756.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Oza PP, Kashfi K. The evolving landscape of PCSK9 inhibition in cancer. Eur J Pharmacol. 2023;949: 175721. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ejphar.2023.175721.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Mei W, Faraj Tabrizi S, Godina C, Lovisa AF, Isaksson K, Jernström H, Tavazoie SF. A commonly inherited human PCSK9 germline variant drives breast cancer metastasis via LRP1 receptor. Cell. 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.cell.2024.11.009.

    Article  PubMed  Google Scholar 

  89. Kosmas CE, Silverio D, Ovalle J, Montan PD, Guzman E. Patient adherence, compliance, and perspectives on evolocumab for the management of resistant hypercholesterolemia. PPA. 2018;12:2263–6. https://doiorg.publicaciones.saludcastillayleon.es/10.2147/PPA.S149423.

    Article  PubMed  PubMed Central  Google Scholar 

  90. Zenti MG, Stefanutti C, Sanga V, Altomari A, Fabris A, Dauriz M, Bonora E. Evolocumab and lipoprotein apheresis combination therapy may have synergic effects to reduce low-density lipoprotein cholesterol levels in heterozygous familial hypercholesterolemia: a case report. J Clin Apheresis. 2018;33:546–50. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/jca.21632.

    Article  CAS  PubMed  Google Scholar 

  91. Zhang Y, Zhang A, Wu Y, Zhang Y, Hu W, Chen P, Chen K, Ding J. Early addition of evolocumab to statin treatment in patients with acute coronary syndrome and multivessel disease undergoing percutaneous coronary intervention. Rev Cardiovasc Med. 2023;24:270. https://doiorg.publicaciones.saludcastillayleon.es/10.31083/j.rcm2409270.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. F. Imarhia. 2021. Evaluation of the Effectiveness and Safety of Alirocumab Use in Statin-Intolerant Veterans. Fed Pract. https://doiorg.publicaciones.saludcastillayleon.es/10.12788/fp.0176.

  93. Quagliariello V, Bisceglia I, Berretta M, Iovine M, Canale ML, Maurea C, Giordano V, Paccone A, Inno A, Maurea N. PCSK9 inhibitors in cancer patients treated with immune-checkpoint inhibitors to reduce cardiovascular events: new frontiers in cardioncology. Cancers. 2023;15:1397. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cancers15051397.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Goksøyr L, Skrzypczak M, Sampson M, Nielsen MA, Salanti A, Theander TG, Remaley AT, De Jongh WA, Sander AF. A cVLP-based vaccine displaying full-length pcsk9 elicits a higher reduction in plasma PCSK9 than similar peptide-based cVLP vaccines. Vaccines. 2022;11:2. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/vaccines11010002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Galabova G, Brunner S, Winsauer G, Juno C, Wanko B, Mairhofer A, Lührs P, Schneeberger A, Von Bonin A, Mattner F, Schmidt W, Staffler G. Peptide-based anti-PCSK9 vaccines—an approach for long-term LDLc management. PLoS ONE. 2014;9: e114469. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0114469.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Toth S, Pella D, Fedacko J. Vaccines Targeting PSCK9 for the Treatment of Hyperlipidemia. Cardiol Ther. 2020;9:323–32. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s40119-020-00191-6.

    Article  PubMed  PubMed Central  Google Scholar 

  97. Momtazi-Borojeni AA, Ebrahimi Nik M, Reza Jaafari M, Banach M, Sahebkar A. Effects of immunization against PCSK9 in an experimental model of breast cancer. Aoms. 2019;15:570–9. https://doiorg.publicaciones.saludcastillayleon.es/10.5114/aoms.2019.84734.

    Article  CAS  Google Scholar 

  98. Wang H, Zhang X, Zhang Y, Shi T, Zhang Y, Song X, Liu B, Wang Y, Wei J. Targeting PCSK9 to upregulate MHC-II on the surface of tumor cells in tumor immunotherapy. BMC Cancer. 2024;24:445. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-024-12148-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Ruzzi F, Semprini MS, Scalambra L, Palladini A, Angelicola S, Cappello C, Pittino OM, Nanni P, Lollini P-L. Virus-like particle (VLP) vaccines for cancer immunotherapy. IJMS. 2023;24:12963. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms241612963.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Sander AF, Lollini P-L. Virus-like antigen display for cancer vaccine development, what is the potential? Expert Rev Vaccines. 2018;17:285–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/14760584.2018.1455505.

    Article  PubMed  Google Scholar 

  101. Tornesello AL, Tagliamonte M, Buonaguro FM, Tornesello ML, Buonaguro L. Virus-like particles as preventive and therapeutic cancer vaccines. Vaccines. 2022;10:227. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/vaccines10020227.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Mohsen MO, Speiser DE, Knuth A, Bachmann MF. Virus-like particles for vaccination against cancer. WIREs Nanomed Nanobiotechnol. 2020;12: e1579. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/wnan.1579.

    Article  CAS  Google Scholar 

  103. Harper DM, Franco EL, Wheeler C, Ferris DG, Jenkins D, Schuind A, Zahaf T, Innis B, Naud P, De Carvalho NS, Roteli-Martins CM, Teixeira J, Blatter MM, Korn AP, Quint W, Dubin G. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. The Lancet. 2004;364:1757–65. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S0140-6736(04)17398-4.

    Article  CAS  Google Scholar 

  104. Cubas R, Zhang S, Li M, Chen C, Yao Q. Chimeric Trop2 virus-like particles: a potential immunotherapeutic approach against pancreatic cancer. J Immunother. 2011;34:251–63. https://doiorg.publicaciones.saludcastillayleon.es/10.1097/CJI.0b013e318209ee72.

    Article  CAS  PubMed  Google Scholar 

  105. Ruzzi F, Palladini A, Clemmensen S, Strøbæk A, Buijs N, Domeyer T, Dorosz J, Soroka V, Grzadziela D, Rasmussen CJ, Nielsen IB, Soegaard M, Semprini MS, Scalambra L, Angelicola S, Landuzzi L, Lollini P-L, Thorn M. Prevention and therapy of metastatic HER-2+ mammary carcinoma with a human candidate HER-2 virus-like particle vaccine. Biomedicines. 2022;10:2654. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/biomedicines10102654.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Hu H, Steinmetz NF. Development of a virus-like particle-based anti-HER2 breast cancer vaccine. Cancers. 2021;13:2909. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/cancers13122909.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Nika L, Cuadrado-Castano S, Asthagiri Arunkumar G, Grünwald-Gruber C, McMahon M, Koczka K, García-Sastre A, Krammer F, Grabherr R. A HER2-displaying virus-like particle vaccine protects from challenge with mammary carcinoma cells in a mouse model. Vaccines. 2019;7:41. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/vaccines7020041.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Razazan A, Nicastro J, Slavcev R, Barati N, Arab A, Mosaffa F, Jaafari MR, Behravan J. Lambda bacteriophage nanoparticles displaying GP2, a HER2/neu derived peptide, induce prophylactic and therapeutic activities against TUBO tumor model in mice. Sci Rep. 2019;9:2221. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41598-018-38371-z.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Ruzzi F, Semprini MS, Scalambra L, Angelicola S, Cappello C, Pittino OM, Nanni P, Palladini A, Lollini P-L. Functional evaluation of circulating anti-cancer antibodies with a 3D tumor cell growth inhibition assay. In: Ruzzi F, editor. Methods in Cell Biology. Amsterdam: Elsevier; 2024. p. 75–86. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/bs.mcb.2024.08.010.

    Chapter  Google Scholar 

  110. Nanni P, Nicoletti G, De Giovanni C, Landuzzi L, Di Carlo E, Cavallo F, Pupa SM, Rossi I, Colombo MP, Ricci C, Astolfi A, Musiani P, Forni G, Lollini P-L. Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice. J Exp Med. 2001;194:1195–206. https://doiorg.publicaciones.saludcastillayleon.es/10.1084/jem.194.9.1195.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Rovero S, Amici A, Carlo ED, Bei R, Nanni P, Quaglino E, Porcedda P, Boggio K, Smorlesi A, Lollini P-L, Landuzzi L, Colombo MP, Giovarelli M, Musiani P, Forni G. DNA vaccination against rat her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. J Immunol. 2000;165:5133–42. https://doiorg.publicaciones.saludcastillayleon.es/10.4049/jimmunol.165.9.5133.

    Article  CAS  PubMed  Google Scholar 

  112. Vidor E. The nature and consequences of intra- and inter-vaccine interference. J Comp Pathol. 2007;137:S62–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jcpa.2007.04.014.

    Article  CAS  PubMed  Google Scholar 

  113. Nanni P, Landuzzi L, Nicoletti G, De Giovanni C, Rossi I, Croci S, Astolfi A, Iezzi M, Di Carlo E, Musiani P, Forni G, Lollini P-L. Immunoprevention of mammary carcinoma in HER-2/ neu transgenic mice is IFN-γ and B cell dependent. J Immunol. 2004;173:2288–96. https://doiorg.publicaciones.saludcastillayleon.es/10.4049/jimmunol.173.4.2288.

    Article  CAS  PubMed  Google Scholar 

  114. Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother. 2005;54:721–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00262-004-0653-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Germann T, Gately MK, Schoenhaut DS, Lohoff M, Mattner F, Fischer S, Jin S, Schmitt E, Rüde E. Interleukin-12/T cell stimulating factor, a cytokine with multiple effects on T helper type 1 (T h 1) but not on T h 2 cells. Eur J Immunol. 1993;23:1762–70. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/eji.1830230805.

    Article  CAS  PubMed  Google Scholar 

  116. Stevens TL, Bossie A, Sanders VM, Fernandez-Botran R, Coffman RL, Mosmann TR, Vitetta ES. Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature. 1988;334:255–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/334255a0.

    Article  CAS  PubMed  Google Scholar 

  117. Pereira PMR, Sharma SK, Carter LM, Edwards KJ, Pourat J, Ragupathi A, Janjigian YY, Durack JC, Lewis JS. Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy. Nat Commun. 2018;9:5137. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41467-018-07608-w.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Yuan J, Cai T, Zheng X, Ren Y, Qi J, Lu X, Chen H, Lin H, Chen Z, Liu M, He S, Chen Q, Feng S, Wu Y, Zhang Z, Ding Y, Yang W. Potentiating CD8+ T cell antitumor activity by inhibiting PCSK9 to promote LDLR-mediated TCR recycling and signaling, protein. Cell. 2021;12:240–60. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s13238-021-00821-2.

    Article  CAS  Google Scholar 

  119. Liu X, Bao X, Hu M, Chang H, Jiao M, Cheng J, Xie L, Huang Q, Li F, Li C-Y. Inhibition of PCSK9 potentiates immune checkpoint therapy for cancer. Nature. 2020;588:693–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41586-020-2911-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Sun S, Yang Z, Yao H, Zhang Z. A new enhancer for anti-PD-1/PD-L1 immunotherapy: PCSK9 inhibition. Trends Cancer. 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.trecan.2024.10.002.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

The Authors with to thanks the people at ExpreS2ion Biotechnologies, Hørsholm, Denmark, for their help with the preparation of proteins for the cVLP vaccines.

Funding

This research received no external funding.

Author information

Author notes

  1. Laura Scalambra and Francesca Ruzzi have contributed equally.

  2. Pier-Luigi Lollini and Adam Frederik Sander jointly supervised this work.

Authors and Affiliations

  1. Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Bologna, Italy

    Laura Scalambra, Francesca Ruzzi, Olga Maria Pittino, Maria Sofia Semprini, Chiara Cappello, Stefania Angelicola, Patrizia Nanni & Pier-Luigi Lollini

  2. IRCCS Policlinico Di Sant’Orsola University Hospital, Bologna, Italy

    Stefania Angelicola & Pier-Luigi Lollini

  3. Department of Molecular Medicine, University of Pavia, Pavia, Italy

    Arianna Palladini

  4. Medical Oncology Division, IRCCS Policlinico San Matteo, Pavia, Italy

    Arianna Palladini

  5. AdaptVac Aps, Copenhagen, Denmark

    Louise Goksøyr, Cyrielle Fougeroux & Adam Frederik Sander

  6. Division of Surgical Oncology, Department of Surgery and Department of Microbiology, Immunology and Molecular GeneticsThe Molecular Biology InstituteJonsson Comprehensive Cancer Centre, University of California, Los Angeles (UCLA), CA, USA

    Manuel L. Penichet

  7. Centre for Translational Medicine and Parasitology, Department for Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Adam Frederik Sander

Authors
  1. Laura Scalambra
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Francesca Ruzzi
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Olga Maria Pittino
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Maria Sofia Semprini
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Chiara Cappello
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Stefania Angelicola
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Arianna Palladini
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Patrizia Nanni
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Louise Goksøyr
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Cyrielle Fougeroux
    View author publications

    You can also search for this author inPubMed Google Scholar

  11. Manuel L. Penichet
    View author publications

    You can also search for this author inPubMed Google Scholar

  12. Adam Frederik Sander
    View author publications

    You can also search for this author inPubMed Google Scholar

  13. Pier-Luigi Lollini
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Conceptualization, P.-L.L. and A.F.S; methodology, F.R., L.S., O.P., C.C., M.S.S., S.A., P.N., A.P., M.P., C.F., L.G., P.-L.L. and A.F.S.; formal analysis, P.-L.L., F.R., L.S. P.N.; investigation,, F.R., L.S., O.P., C.C., M.S.S., S.A., P.N., A.P., C.F., L.G., P.-L.L. and A.F.S.; resources, F.R., L.S., O.P., C.C., M.S.S., S.A., P.N., A.P., M.P., C.F., L.G., P.-L.L. and A.F.S.; data curation, P.-L.L., F.R., L.S., P.N.; writing—original draft preparation, P.-L.L., F.R. and L.S.; writing—review and editing, all authors; supervision, P.-L.L. and A.F.S; project administration, P.-L.L. and A.F.S. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Francesca Ruzzi or Pier-Luigi Lollini.

Ethics declarations

Ethics approval and consent to participate

All in vivo experiments were performed according to Italian and European laws and were authorized by the Italian Ministry of Health (letter 714–2017-PR).

Consent for Publication

Not applicable.

Competing interests

L.G., C.F. and A.F.S. are listed as co-inventors on a patent application covering the Delivery of a cVLP-based modular vaccine platform in a nucleic acid platform (P5856PC00). Employees of AdaptVac (L.G., C.F. and A.F.S.), a company commercializing virus-like particle display technology and vaccine, including several patents.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Scalambra, L., Ruzzi, F., Pittino, O.M. et al. Targeting PCSK9, through an innovative cVLP-based vaccine, enhanced the therapeutic activity of a cVLP-HER2 vaccine in a preclinical model of HER2-positive mammary carcinoma. J Transl Med 23, 136 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06126-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06126-w

Keywords

Journal of Translational Medicine

ISSN: 1479-5876

Contact us